TPEN

Zinc chelator TPEN induces pancreatic cancer cell death through causing oxidative stress and inhibiting cell autophagy

Zhen Yu1* | Ze Yu1* | ZhenBao Chen1 | Lin Yang1 | MingJun Ma1 |
ShouNan Lu2 | ChunSheng Wang1 | ChunBo Teng1 | YuZhe Nie1

1College of Life Science, Northeast Forestry University, Harbin, China
2Department of Hepatopancreatobiliary Surgery, Second Affiliated Hospital of Harbin Medical University, Harbin, China

Correspondence

Chun‐Bo Teng and Yu Zhe Nie, Hexing Road 26, 150040 Harbin, China. Email: [email protected] (C. B. Teng); [email protected] (Y. Z. Nie)

Funding information

National Natural Science Foundation of China, Grant/Award Number: 31472159; Fundamental Research Funds for the Central Universities of China, Grant/Award Numbers: 2572016AA14, 2572016EAJ3; Natural Science Foundation of Heilongjiang Province, Grant/Award Number: ZD2017001

1 | INTRODUCTION

Zinc is an essential trace element and catalytic cofactor involved in cell growth and organism development (Prasad, 2013). Alterations of zinc concentration in cells impact many physiological processes (Hoang, Han, Shaw, & Nimni, 2016; Rudolf & Rudolf, 2015), mainly through its signaling, structural, regulatory, and catalytic functions (Haase & Rink, 2014; Kulikova, Makarov, & Kozin, 2015; Sun et al., 2018). With regard to its catalytic functions, this trace element is intimately involved in the function of over 300 enzymes (Jen & Wang, 2016). Supplementation of zinc has been shown to promote DNA synthesis, whereas depletion of this mineral inhibits DNA synthesis (Gening, Lakhin, Stelmashook, Isaev, & Tarantul, 2013).

Zinc aberrations also involves closely with cellular dysfunction and cancer progression. Some evidence demonstrated that zinc aberrations could lead to DNA damage and the initiation of cancer (Dhawan & Chadha, 2010; Ho, 2004). Zinc is important for tumor growth. Evidence in support of this idea is extensive (Kocdor et al., 2015; Singh, Pitschmann, & Ahmad, 2014; Supasai, Aimo, Adamo, Mackenzie, & Oteiza, 2017). Besides, in vivo experiment revealed paternal zinc deficiency in rats acted as an important factor in pup cancer, through increasing oxidative damage of DNA in testicular cells (Maremanda, Khan, & Jena, 2016).

Pancreatic cancer has the lowest overall survival rate in all cancers. Its 5‐year survival rate is less than 5% (Tang & Chen, 2014). However, very little is known about the molecular mechanism of pancreatic cancer. Therefore, novel therapeutic targets are urgently needed for this terrible cancer. The deregulated zinc transport (such as ZIP4) in pancreatic cancer might provide new insights on tumor growth and metabolism (Cui et al., 2014; Li et al., 2009). Overexpression of zinc transporters may regulate the expression of oncogenes through providing zinc to the tumor cells. On the other hand, cell autophagy has recently emerged as a significant mechanism in pancreatic cancer development. Lots of studies indicate that autophagy has a cytoprotec- tive effect, and it is required for pancreatic cancer growth (Seo et al., 2016; S. Yang et al., 2011; A. Yang et al., 2014). Simultaneously, abundant evidence supports that zinc is a positive regulator of autophagy (Lee & Koh, 2010; Liuzzi, Guo, Yoo, & Stewart, 2014). Affluent zinc content in medium has been shown to enhance autophagy induced by tamoxifen in retinal pigment epithelial and breast cancer cells (Cho, Yoon, Choi, Lee, & Koh, 2012; Hwang et al., 2010).

Previous reports have shown both an increase and a decrease of intracellular zinc are associated with cancer development. The purpose of our work is to explore the effect of depleting endogenous zinc, by using the zinc chelator N,N,N,N‐Tetrakis(2‐pyridylmethyl)-ethylenediamine (TPEN), which is a lipid‐soluble metal chelator and has been shown to induce apoptosis in several cancer cells (Pang et al., 2013; Seth et al., 2015).

It is reported that TPEN could induce apoptosis of pancreatic cancer cells CFPAC1, PaCa44, and HPAF II. The cell apoptosis induced by TPEN was involved in caspase‐3/8 activation (Donadelli et al., 2008). However, the molecular mechanism of cell death signaling induced by TPEN in pancreatic cancer cells has not been adequately established. In the present study, we sought to identify the potential roles of both mitochondrial dysfunction and autophagy destroy in TPEN‐induced pancreatic cancer cell death.

2 | MATERIALS AND METHODS

2.1 | Patients and clinical specimens

Human pancreatic cancer samples were obtained from 16 patients after surgical resection at the Second Affiliated Hospital of Harbin Medical University in Harbin, China. Informed consent was obtained from the patients, and the research was approved by the appropriate committees of the Harbin Medical University. None of the patients had received neoadjuvant chemotherapy. Fresh frozen samples were obtained for inductively coupled plasma‐mass spectrometry (ICP‐MS) metal element content analysis.

2.2 | ICP‐MS assay

The measurement of elements was determined by a 7500 cx Agilent inductively coupled plasma mass spectrometry instrument (Agilent, Santa Clara, CA). All tissue samples were dried for 72 hr at 60°C. Zinc content assays were running in the reaction mode with high‐purity ammonia as the reaction cell gas. The sample introduction system consisted of a quartz cyclonic spray chamber and a glass Type C nebulizer.

2.3 | Cell culture

The human pancreatic cancer cell lines Panc‐1, BxPc‐3, 8988T, and L3.6; human cervical carcinoma cell line Hela; human breast cancer cell line MDA‐MB‐231, human malignant glioma cell line U87; and human liver cancer cell line HepG2 were obtained from the American Type Culture Collection (Chicago, IL) and cultured in a complete growth medium. The cultured cells were maintained in a 5% CO2 concentration at 37°C. All the cancer cells were cultured in RPMI‐ 1640 medium supplemented with 10% fetal bovine serum (FBS), 100 μg/ml ampicillin, 100 μg/ml kanamycin (Sigma, St. Louis, MO), and 2 mM L‐glutamine.

For the pancreatic cancer cell sphere culture, Panc‐1 cells in RPMI 1640 medium with 0.25% Matrigel were plated into a prepared 96‐well plate with 2% agarose in RPMI 1640 medium (5 cells per well) and cultured for 3–9 days.For treatments, cells were treated with various reagents, including TPEN, a membrane‐permeable zinc chelator; N‐acetylcys- teine (NAC), an antioxidant compound, and chloroquine diphosphate salt (CQ), an inhibitor of late phase of autophagy. All the reagents were provided by Sigma. Final concentrations and incubation times for all experiments are reported in the figure legends.

2.4 | Cell proliferation assay

Cell proliferation was examined using the cell counting Kit‐8 (CCK‐8; Beyotime, Shanghai, China) assay according to the instructions from the manufacturer. Briefly, Panc‐1, BxPc‐3, 8988T, L3.6, Hela, MDA‐MB‐231, U87, and HepG2 cells were placed in a 96‐well plate at a density of 5,000 cells/well, respectively. Cells were allowed to adhere for 24 hr at 37°C and then treated with various inhibitors (TPEN or
CQ). Afterward, the supernatants were removed and replaced with 100 μl of fresh medium containing 10 μl of CCK‐8 solution and the cells were incubated for 2 hr at 37°C in the dark. Immediately after the incubation, the optical density of each well at 450 nm (OD value) was measured with a microplate Reader (TECAN, Kawasaki, Japan). Cell viability was expressed as percentage absorbance of cells treated with inhibitors compared with the percentage absorbance of untreated cells.

2.5 | Measurement of cell death by flow cytometry

Cell death was measured by flow cytometry using propidium iodide (PI) (Vazyme Biotech, Nanjing, China) stain for TPEN cytotoxic experiments. Diverse cancer cells, treated with different doses of TPEN, were kept under stress conditions for 24 hr before the cell death assay. These cancer cells were harvested and washed once in cold phosphate‐buffered saline (PBS), and then stained with PI for 30 min at room temperature in the dark. After staining, the cells were analyzed by flow cytometry using 488 nm excitation. The percentage of death cells corresponds to PI‐ positive cells. All samples were analyzed in a flow cytometry (BD C6 Biosciences, San Jose, CA).

2.6 | Wound healing assay

For the wound healing assays, Panc‐1 cells treated with different doses of TPEN were plated in 24‐well culture plates. The monolayer
cells were scratched using a small pipette tip, washed once with serum‐free medium, and then were cultured in RPMI‐1640 medium with 2% FBS. After 48 hr, migration was assessed microscopically.

2.7 | Cell invasion assay

Cell invasion was tested using a transwell assay system (Corning, NY). The TPEN‐treated Panc‐1 cells were seeded with serum‐free
RPMI‐1640 medium and plated into the upper layer polycarbonate membrane filter; RPMI‐1640 medium with 10% FBS was added to the bottom chambers. After 72 hr, the cells that crossed upper layer polycarbonate to the bottom chambers were fixed with 4% paraformaldehyde (PFA), stained with 0.05% crystal violet, and counted. The data presented are the mean ± standard error (SE) and represent three independent experiments.

2.8 | Colony formation assay

Panc‐1 cells were plated into 12‐well culture plates at a concentra- tion of 200 cells per plate. Then cells were exposed to different doses of TPEN for 12 days. All colonies were fixed in 4% PFA for 15 min, stained with 0.05% crystal violet, and counted. The data presented are the mean ± SE and represent three independent experiments.

2.9 | Measurement of intracellular ROS levels

Intracellular reactive oxygen species (ROS) generation was assessed using the peroxide‐sensitive fluorescent probe 2′,7′‐dichlorofluorescin diacetate (DCFH‐DA; Beyotime) in accordance with the instructions. After collection, cells were incubated with the dye of DCFH‐DA, which was attenuated with serum‐free Dulbecco’s modified Eagle’s medium (DMEM) at a proportion of 1:1,000, for 30 min at 37°C in the dark,washed twice with PBS, and then resuspended in PBS to detect the generation of intracellular ROS by flow cytometry (BD C6 Biosciences). In all experiments, 20,000 viable cells were analyzed and data analysis was performed by using Flow Jo software. Simultaneously, cell images were taken using a fluorescence microscope for magnifications ×40 and Delta Vision Imaging Workstation (×60), and the fluorescence analysis was performed using the ImageJ software.

2.10 | Determination of GSH and GSSG

The intracellular glutathione (GSH) and glutathione disulfide (GSSG) level was measured by GSH and GSSG Assay Kit (S0053) from Beyotime. Panc‐1 cells were treated with TPEN for different hours, collected and then lyzed by releasing buffer on ice. According to the
protocols of the manufacturer, the standard curve of the absorbance to GSH and GSSG concentrations was measured. Then, we determined the GSH and GSSG concentration using a microplate reader at 412 nm.

2.11 | Measurement of mitochondrial membrane potential

Intracellular mitochondrial membrane potential (MMP) was assessed using the Rhodamine123, an orange fluorescent dye which is sensitive to MMP (C2007; Beyotime). According to the instructions, cell images were taken using fluorescence microscope for magnifica- tions ×10 and ×40, Delta Vision Imaging Workstation (×60), and the images analysis were performed using the image J‐MiNA software (Sanderson, Reynolds, Kumar, Przyklenk, & Hüttemann, 2013).

2.12 | ATP production assay

The intracellular adenosine triphosphate (ATP) level was measured by the ATP Assay Kit (S0053, Beyotime). Panc‐1 cells were treated with TPEN for different hours, collected and then lyzed by ATP releasing buffer on ice. According to the protocols, the standard curve of the absorbance to ATP concentrations was measured. Then luciferase activity was determined after 24 hr and 48 hr using the GloMax‐20/20 Luminometer from Promega.

2.13 | Measurement of intracellular pH

Intracellular pH was detected using the penetrate‐membrane fluorescent probe 3’‐O‐Acetyl‐2′,7’‐bis(carboxyethyl)‐4 or 5‐carbox- yfluorescein, diacetoxymethyl ester (BCECF‐AM), provided by Beyotime. In accordance with the instructions, the BCECF‐AM probe was attenuated with serum‐free DMEM at a proportion of 1:1,000, for 40 min at 37°C in the dark. Cell images were taken using the inverted fluorescence microscope for magnifications ×40, and the fluorescence analysis was performed using the ImageJ software.

2.14 | Lactate dehydrogenase activity assay

Intracellular total lactate dehydrogenase (LDH) activity was measured by the LDH Assay Kit (C0016; Beyotime). Its principle was an INT chromogenic reaction catalyzed by diaphorase; total LDH activity in cells was detected by colorimetry. According to the protocols of the manufacturer, after collection, cells were incubated with LDH release reagent, and then the optical density of each well at 490 nm (OD value) was measured with a microplate Reader (sunrise; TECAN).

2.15 | Real‐time polymerase chain reaction

Total RNA was extracted using the TRIzol RNA isolation system (Takara, Dalian, China) according to the manufacturer’s instructions. Real‐time polymerase chain reaction (PCR) was performed using SYBR Green Master (Cat#04913914001; Roche, Basel, Switzerland) in a Light‐Cycler 480 System (Roche) used for determination of messenger RNA (mRNA) levels. The following conditions were used for PCR: 95°C for 30 s followed by 40 amplification cycles at 95°C for 5 s and 60°C for 30 s. β‐Actin was used as the internal normalization control. The primer sequences used are listed in Supplementary Material (Table S1). The relative gene expression levels were quantified by normalization to endogenous β‐actin expression levels, which were calculated by the 2−ΔΔC(t) method. All the abbreviations of genes were seen in Supplementary Material.

2.16 | Western blot analysis

The cell lysate was prepared using radio immunoprecipitation assay (RIPA) buffer containing protease inhibitors, phosphatase inhibitors, and dithiothreitol. Protein extracts from Panc‐1 cells were prepared and protein concentration was measured using a BCA protein assay kit (Beyotime). Western blot analyses were performed with the use of specific antibody for LC3A/B (Catalog# 4108) from Cell Signaling Technology (Beverly, MA), β‐actin (A5441) from Sigma Aldrich, and goat antirabbit IgG‐horseradish peroxidase from Cell Signaling Technology.Relative quantification of protein levels was determined by measuring the intensity of the protein bands with the use of the ImageJ software.

2.17 | Imaging mCherry‐GFP‐LC3

Ad‐mCherry‐GFP‐LC3 adenovirus was used to demonstrate autophagy flux in cells (Ma et al., 2017). Panc‐1 cells were grown on 24‐well plates and reached 60–70% confluence before transfection. Cells were transfected with the Ad‐mCherry‐GFP‐LC3 adenovirus at a 50 MOI in 200 μL DMEM containing 10% FBS for 24 hr at 37°C. Next, we removed the culture medium contain- ing the virus, replaced the fresh culture medium, and continued to culture the cells for 48 hours. Cells were visualized by fluores- cence microscopy. MCherry‐LC3 positive cells were located in lysosomes, because the green fluorescent protein (GFP) quenched signal was detected in lysosomes while mCherry‐GFP‐LC3‐positive cells were accumulated in autophagosomes. Autophagy flux was evaluated by calculating the number of yellow and red puncta.

2.18 | Lysosomal staining

Lysosomal activity was performed using the lysosomotropic probe, Lyso‐Tracker Red, which can be selectively retained in the slightly acidic lysosomes. In accordance with the instructions, cells were incubated with the Lyso‐Tracker, which was attenuated with serum‐ free DMEM at a proportion of 1:1,000, for 40 min at 37°C in the dark, washed twice with PBS, and then resuspended in serum‐free DMEM to detect the red fluorescence by Inverted fluorescence microscope. Cell images were taken with magnifications ×10 and ×40, and stained lysosomes were visualized by the ImageJ software. Lysosomes can also be stained with 0.1% neutral red at room temperature and appeared red under a common inverted micro- scope.

FIG U RE 1 Most SLC39A family members are upregulated in PDAC. (a) Differential expression levels of SLC39A family in breast tumor and normal tissues and the diagrammatic sketch of intracellular localization of ZIPs and ZnTs. (b) ZIPs and ZnTs are differentially expressed in the pancreatic tumor samples. (c) IHC staining of SLC39A (ZIP10) was performed in PDAC and normal tissues. (d) ICP‐MS was used to detect the contents of zinc element in the 16 groups of tissue samples. IHC: immunohistochemistry; ICP‐MS: inductively coupled plasma‐mass
spectrometry; PDAC: pancreatic ductal adenocarcinoma [Color figure can be viewed at wileyonlinelibrary.com].

FIG U RE 2 TPEN induces pancreatic cancer cell death. (a) Four kinds of pancreatic cancer cell lines (Panc‐1, BxPc‐3, 8988T, and L3.6) were treated with 0–6 µM TPEN for 24 hr and cell viability (proliferation) was assessed by the CCK‐8 assay. (b) Representative microscopic images of various concentrations of TPEN treatment groups in Panc‐1 cells after 24 hr. (c) The ratio of PI‐positive cells was significantly and dose dependently increased by TPEN through flow cytometry. (d) At 24 hr of incubation after the 4 µM TPEN treatment, flow cytometry showed that the ratio of PI‐positive cells was significantly higher in Panc‐1 cells than in other cell lines (Hela, MDA‐MB‐231, U87, and HepG2). All the results are presented as the means ± SEM of values obtained in three independent experiments, *p < 0.05, **p < 0.01, ***p < 0.001 (Student’s t‐test). PI: propidium iodide; SEM: standard error of mean; TPEN: N,N,N,N‐Tetrakis(2‐pyridylmethyl)‐ethylenediamine [Color figure can be viewed at wileyonlinelibrary.com]. 2.19 | Bioinformatics analyses All bioinformatics analysis methods and websites were shown in Supplementary Material. 2.20 | Statistical analysis All the bars or symbols in the graph represent the means ± standard deviation error from at least three independent experiments with similar results. The results were analyzed by Student’s t‐test and in all analysis, *p < 0.05, **p < 0.01, and ***p < 0.001 were considered statistically significant. 3 | RESULTS 3.1 | The concentration of zinc is high in pancreatic cancer In mammalian cells, there are two zinc transporter protein families, SLC39A family (ZIPs) and SLC30A family (ZnTs), which have opposite roles in maintaining cellular zinc homeostasis: ZIP transporters are responsible for zinc uptake, whereas ZnTs prompt zinc efflux. There are 14 ZIPs and 10 ZnTs proteins identified in human, with tissue‐specific expression (J. Yang et al., 2013). According to the relevant research works, ZIP1–6, ZIP8, ZIP10, and ZIP14 are responsible for zinc uptake (Lee et al., 2015). Based on the above, we speculated that the expression level of these ZIPs in pancreatic cancer could indicate the concentration of zinc in cells. As shown in Figure 1a, we analyzed the data from the gene expression profiling interactive analysis (GEPIA) site based on TCGA (Tang et al., 2017). In addition to ZIP5 and ZIP14, the levels of other ZIPs mentioned above were higher in pancreatic cancer than those in normal tissues. Meanwhile, according to the data of National Coalition Building Institute (NCBI), gene expression omnibus (GEO) Datasets (GSE16515) (Pei et al., 2009), comparing the background levels of these kinds of ZIPs and ZnTs, it was seen that the mRNA level of ZIP10 was the highest in the chip data of pancreatic cancer (Figure 1b), as the same as what ZIP10 protein level displayed (Liu & Liu, 2015; Figure 1c). Based on these findings, we speculated that the concentration of zinc in pancreatic cancer should be higher than those in normal tissues. We used ICP‐MS analysis to determine the metal content of zinc in the tissue samples from pancreatic cancer patients. The results showed that zinc content in tumor tissues was higher than that in adjacent non‐cancer tissues (Figure 1d). These results indicated that high concentration of zinc was beneficial to the occurrence and development of pancreatic cancer. FIG U RE 3 TPEN inhibits pancreatic cancer progression. (a) The effect of 4 µM TPEN on cell viability as measured by CCK‐8 assay. ZnSO4 can completely reverse this phenotype and CuSO4 can partly reverse this phenotype. (b) The growth of Panc‐1 cell spheres was inhibited by TPEN. TPEN (4 µM) was added on the 6th day after the cells were plated and the difference of cell spheres treated with TPEN was observed after 3 days, compared with control DMSO. The results are presented as the means ± SEM of values obtained in three independent cell spheres, ***p < 0.001 (Student’s t‐test). (c) Wound healing assay of panc‐1 cells treated with different doses of TPEN. Representative pictures were shown at 0 hr and 48 hr after the wound was made. (d) Transwell assay of panc‐1 cells treated with different doses of TPEN represented the cell invasive ability. (e) Colony formation assay of panc‐1 cells treated with different doses of TPEN. DMSO: dimethyl sulfoxide; SEM: standard error of mean; TPEN: N,N,N,N‐Tetrakis(2‐pyridylmethyl)‐ethylenediamine [Color figure can be viewed at wileyonlinelibrary.com]. FIG U RE 4 TPEN induces oxidative stress in Panc‐1 cells. (a) The ROS levels of Panc‐1 cells treated with 4 µM TPEN was measured by DCFH‐DA probe fluorescence detection. (b) The effect of 4 µM or 6 µM TPEN on cell viability as measured by the CCK‐8 assay. ZnSO4 can completely reverse these phenotypes and NAC can partly reverse these phenotypes. (c) The ROS levels of different cancer cells treated with 4 µM TPEN were measured by flow cytometry. (d) The ROS levels of Panc‐1 cells treated with 4 µM TPEN were captured with a Delta Vision Imaging Workstation every 4 hr. (e) TPEN‐ induced ROS elevation accompanied by changing total GSH and GSSG level in Panc‐1 cells within 16 hr. DMSO was used as the negative control in all TPEN treatment. All the results are presented as the means ± SEM of values obtained in three independent experiments, *p < 0.05, **p < 0.01, ***p < 0.001 (Student’s t‐test). DCFH‐DA: 2′,7′‐dichlorofluorescin diacetate; DMSO: dimethyl sulfoxide; GSH: glutathione; GSSG: glutathione disulfide; NAC: N‐acetylcysteine; ROS: reactive oxygen species; SEM: standard error of mean; TPEN: N,N,N,N‐Tetrakis(2‐pyridylmethyl)‐ethylenediamine [Color figure can be viewed at wileyonlinelibrary.com]. 3.2 | TPEN triggers pancreatic cancer cell death in a dose‐dependent manner We found that TPEN was cytotoxic to pancreatic cancer cells (Panc‐1, BxPc‐3, 8988T, and L3.6) in a dose‐dependent way using CCK8 assay (Figure 2a). Exposure to 4 µM TPEN for 24 hr was enough to induce a reduction of approximate 50% in cell viability. And almost 80% inhibition of the proliferation of Panc‐1 and 8988T cells was achieved when incubated with 6 µM TPEN for 24 hr. These data suggested that TPEN induces growth arrest of pancreatic cancer cells in a dose‐dependent manner. We further observed cell morphology of these four kinds of cells treated with TPEN, and we can see that both 4 µM and 6 µM TPEN cause cell death (Figures 2b and S1A). Typical apoptotic morphology of Panc‐1 cells, apoptotic bodies were also observed following treatment with 4 µM TPEN for 24 hr (Figure 2b). And according to the results of DNA ladder, the degree of apoptosis induced by 4 µM and 6 µM TPEN was almost the same (Figure S1B). On the other hand, we used 4',6‐ diamidino‐2‐phenylindole (DAPI) to stain the nuclei treated with TPEN and it was observed that TPEN induced fragmentation of nuclei (Figure S1C). Next, we detected cell death using PI staining for flow cytometry (Figure 2c). To verify the sensitivity of cytotoxicity treated with TPEN to other cancer cells, we used 4 µM TPEN to treat Hela, MDA‐MB‐231, U87, and HepG2 cells, and these results are shown in Figure 2d, which revealed that cytotoxicity of 4 µM TPEN to Panc‐1 cells was significantly higher than that to other cancer cell lines. 3.3 | TPEN inhibits the progression of pancreatic cancer The above results proved that TPEN significantly caused apoptosis. Next, we will explain whether this apoptosis phenotype is caused by zinc deficiency. First, we added TPEN into the culture medium for 12 hr, and then changed the fresh culture medium. After replacing the medium, we added a certain concentration of zinc sulfate and detected the cell vitality 24 hr later. The rescue test illustrated zinc sulfate could completely recover the cell death of Panc‐1 cells treated with TPEN (Figure 3a). At the same time, it was documented that TPEN is one such metal chelator not only for complexes with zinc, but also for copper to suppress tumor growth (Hyun et al., 2001). Therefore, we performed the same rescue experiment with copper sulfate, and the results showed that copper sulfate could also partially recover the phenotype of cell death treated with TPEN (Figure 3a), yet the rescue effect of copper is far less than that of zinc. FIG U RE 5 TPEN destroys mitochondrial function. (a) TPEN‐induced increase in mitochondrial membrane potential via Rhodamine123 green fluorescence. (b) Rhodamine123 green fluorescence showed more diffuse in Panc‐1 cells treated with TPEN. (c) Visualization and quantitative analysis of diffusion degree in Panc‐1 cells treated with TPEN through the image J‐MiNA software. (d) Different concentrations of TPEN‐induced reduction of ATP in Panc‐1 cells after 24 hr and 48 hr. (e) The ATP ratios of Panc‐1 cells treated with 2 µM or 4 µM TPEN were measured by the GloMax‐20/20 Luminometer every 1 hr. DMSO was used as the negative control in all TPEN treatment. All the results are presented as the means ± SEM of values obtained in three independent experiments, *p < 0.05, ***p < 0.001 (Student’s t‐test). ATP: adenosine triphosphate; DMSO: dimethyl sulfoxide; SEM: standard error of mean; TPEN: N,N,N,N‐Tetrakis(2‐pyridylmethyl)‐ethylenediamine [Color figure can be viewed at wileyonlinelibrary.com]. Besides, we also tested the capabilities of Panc‐1 cells treated with TPEN for cell spheroidization, migration, invasion, and colony formation, and found that TPEN not only induced pancreatic cancer cell death, but also inhibited cell sphere formation efficiency (Figure 3b), cell migration (Figure 3c), invasion (Figure 3d), and colony formation (Figure 3e) in a dose‐ dependent manner. These results suggested that TPEN significantly inhibited the progression of pancreatic cancer. 3.4 | TPEN induces oxidative stress in pancreatic cancer cells Oxidative stress plays important roles in regulating apoptosis of cancer cells (Soliman & Van Dross, 2016; Tor et al., 2014). Therefore, we investigated the generation of ROS treated with TPEN by using fluorescent probe DCFH‐DA. TPEN significantly increased the fluorescence intensity in Panc‐1 cells (Figure 4a). The rescue test illustrated NAC could partially recover the cell death of Panc‐1 cells treated with TPEN (Figure 4b). And the increased ROS level in Panc‐ 1 cells was rather than other cancer cells (Hela, MDA‐MB‐231, U87, and HepG2) (Figure 4c). Next, we intended to explore the changes of ROS levels induced by different TPEN‐incubating time. DCFH‐DA still acting as the ROS probe, Panc‐1 cells images were captured with a Delta Vision Imaging Workstation every 4 hr. Experimental results showed that, with the increase of incubating time, there was no significant change in the ROS level in the control group, yet the ROS level in the TPEN group increased gradually, reached its peak after 12‐hr incubation, and then began to decrease (Figure 4d and S2). At the same time, we also detected the changes of intracellular GSH and GSSG levels, and the GSH/GSSG ratio also reached the bottom at the 12th hour (Figure 4e). The above results indicated that the ROS level of Panc‐1 cells treated with TPEN increased significantly in a time‐dependent way within 12 hr and then began to fall. 3.5 | TPEN destroys mitochondrial function MMP is critical to tricarboxylic acid cycle (TCA) and oxidative phosphorylation (OXPHOS). Strikingly, cells with complete or partial loss of MMP were unable to maintain mitochondrial morphology (Sanderson et al., 2013). Therefore, the maintenance of MMP is an important factor supporting mitochondrial function. We used Rhoda- mine123 to stain mitochondrion in Panc‐1 cells treated with TPEN and found that the fluorescence intensity of the cells increased significantly (Figure 5a). Rhodamine123 is an orange fluorescent dye and sensitive to MMP. If the MMP declines, Rhodamine123 will be released from mitochondrion and diffused in cytoplasm, resulting in an increase in the overall fluorescence intensity of the cell (Figure 5b). To visualize and quantify the dispersion of Rhodamine123, we used the Delta Vision Imaging Workstation to take the cell images and the image J‐MiNA software to process the distribution of Rhodamine123 in cells (Valente, Maddalena, Robb, Moradi, & Stuart, 2017). Statistical analysis showed that the distribution of Rhodamine123 in the cellular network and individual were significantly decreased in Panc‐1 cells treated with TPEN (Figure 5c). Meanwhile, we found that the level of ATP decreased signifi- cantly in Panc‐1 cells treated with TPEN in a dose‐dependent way (Figure 5d). The ATP level in TPEN‐treated Panc‐1 cells treated with TPEN was detected every 1 hr. Compared with 2 µM TPEN‐treated groups, ATP suddenly plummeted in the cells treated with 4 µM TPEN at the 12th hours (Figure 5e). These results illustrated that TPEN could destroy MMP and lead to mitochondrial dysfunction. 3.6 | TPEN induces upregulation of metabolism‐related genes Our previous experiments had shown that TPEN caused cancer cell death by inducing the production of a great deal ROS. In addition to the decrease of the GSH level as previously proved (Figure 4e), there might be other causes for the redox imbalance. We analyzed the data of NCBI: GEO Datasets (GSE49657), which was about the induction of target genes in response to zinc depletion by TPEN and serum starvation in HeLa cells (Homma et al., 2013). Through the gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis, we found that most of the upregulated genes were involved in cholesterol metabolism and transport (Figures S3A and S3B). Cholesterol synthesis needs acetyl‐CoA, which is the metabolism product of glycolysis and fatty acid β‐oxidation. Besides, the TCA cycle that occurs in the mitochondrion also requires acetyl‐CoA. Therefore, we intended to explore whether TPEN could affect glycolysis and fatty acid β‐oxidation in cells. Interestingly, after extracting the target genes related to glucose and fatty acid metabolism from the GEO Datasets, we found the most genes of TCA cycle and glutamic metabolism were upregulated, whereas some genes related to glycolysis and fatty acid β‐oxidation were invariant or downregulated (Figure 6a and 6b). These results implicated that TPEN may affected the mitochondrial metabolism in cancer cells. Then, we detected the expression of metabolism‐related genes in Panc‐1 cells treated with 2 µM or 4 µM TPEN for 9 and 12 hr. As shown in Figure S4 and Figure 6c, 4 µM TPEN could significantly increase the expression levels of glycolysis and TCA cycle related genes, whereas 2 µM TPEN just slightly upregulated the ones. These results implied an increase of glucose metabolism in Panc‐1 cells treated with TPEN within 12 hr. Meanwhile, we also detected pH value in TPEN‐treated Panc‐1 cells in 16 hr (Figure 6d). Increased intracellular acidity reflected upregulation of lactate metabolism. FIG U RE 6 TPEN upregulates metabolism‐related genes. (a,b) The relative expression fold of metabolism‐related genes in Hela cells treated with 8 µM TPEN for 9 hr was determined by gene expression microarray analysis, and the data came from GEO Datasets (GSE49657). (c) The relative expression of metabolism‐related genes in Panc‐1 cells treated with different doses of TPEN for 12 hr was detected by qPCR, and the results were presented as heat‐map. (d) The intracellular pH of Panc‐1 cells treated with 4 µM TPEN for 24 hr was measured by BCECF‐AM probe fluorescence detection. (e) The relative LDH activity of Panc‐1 cells treated with different doses of TPEN for 12 hr was measured with a microplate reader. All the results are presented as the means ± SEM of values obtained in three independent experiments, *p < 0.05, **p < 0.01, ***p < 0.001 (Student’s t‐test). BCECF‐AM: 3'‐O‐Acetyl‐2',7'‐bis(carboxyethyl)‐4 or 5‐carboxyfluorescein, diacetoxymethyl ester; GEO: gene expression omnibus; LDH: lactate dehydrogenase; qPCR: quantitative polymerase chain reaction; SEM: standard error of mean; TPEN: N,N,N,N‐Tetrakis(2‐pyridylmethyl)‐ethylenediamine [Color figure can be viewed at wileyonlinelibrary.com]. Consistent with this finding, lactate dehydrogenase activity raised obviously in Panc‐1 cells after a treatment with 4 µm TPEN for 12 hr (Figure 6e).Increased glycolysis provides enough materials for subsequent mitochondrial metabolism, enhancing OXPHOS, and then producing more ROS. Indeed, an extremely active on mitochondrial respiratory chain is often linked to the increase of the cellular ROS level in various cancers, because the mitochondrial ROS is proportionally correlated to the respiratory activity (Quinlan, Perevoshchikova,Hey‐Mogensen, Orr, & Brand, 2013; Vinogradov & Grivennikova, 2016). Therefore, we presumed TPEN promoted the expression of genes involved in glycolysis, TCA cycle and may contribute to subsequent OXPHOS in a short time, accompanying with a great quantity of generation of by‐products ROS and decreasing GSH level, and finally resulted in ROS accumulation and cell death. 3.7 | TPEN inhibits cell autophagy According to the previous results, NAC can only partially recover TPEN‐induced cell death (Figure 4b). Therefore, we speculated that there were other pathways involved in the cytotoxicity of TPEN. As we mentioned above, zinc is a regulator of autophagy (Cho et al., 2012; Hwang et al., 2010; Lee & Koh, 2010; Liuzzi et al., 2014), and autophagy has been ascribed some roles in both cell survival and cell death, especially in pancreatic cancer cells. Accurate assessment of autophagy is involved in not only the detection of autophagosome, but also the smooth of autophagy flux (Dupont, Orhon, Bauvy, & Codogno, 2014). CQ inhibits the autophagic flux in cancer cells by depressing the lysosomal activity (Chen et al., 2014). In this study, we speculated that the inhibitory effect of TPEN on autophagy was similar with CQ. First, we used mCherry‐LC3‐GFP reporter to detect autophagic flux. As shown in Figure 7a, the fluorescence intensity of GFP and the proportion of GFP positive cells in TPEN‐treated Panc1 cells were significantly higher than those of the control group. This result suggested that TPEN inhibited autophagy flow by depressing lysosomal activity. Next, western‐blot analyses showed TPEN significantly increased the ratio of LC3Ⅱ/Ⅰ (Figure 7b), and it was indicated an increase of early autophagy in the Panc‐1 cells treated with TPEN. To confirm this conclusion, we used the lysosotracker to detect lysosomal activity in Panc‐1 cells treated with TPEN and the results showed that TPEN significantly inhibited lysosomal activity, which was similar to that of the positive control group treated with CQ (Figure 7c). 4 | DISCUSSION Zinc plays dual roles in regulating cell proliferation and apoptosis. Apoptosis induced by zinc depletion is recognized as a potential therapeutic strategy for cancer (Cui et al., 2014; Li et al., 2009). The apoptotic effects of zinc chelator TPEN have been reported on several types of cells, including colon cancer cells and acute promyelocytic leukemia cells (Gurusamy et al., 2011; Rahal et al.,2016; Zhu et al., 2017). The mechanisms of TPEN cytotoxicity are diverse in different tumor cell lines. It was reported that TPEN induced apoptosis in colon cancer cells through inducing ROS production and DNA damage (Fatfat et al., 2014; Rahal et al., 2016). However, in acute lymphoblastic leukemia cells, TPEN‐ induced cell apoptosis was not correlated with its zinc chelation activity. FIG U RE 7 TPEN inhibits autophagy flow by depressing lysosomal activity. (a) mCherry‐LC3‐GFP fluorescence was used to detect TPEN‐induced changes in the level of autophagy, red is on behalf of the early stage of autophagy, green quenching indicates increased lysosomal activity and autophagy flux is unobstructed. And green enhancement indicates lysosomal disruption. TPEN inhibited lysosomal activity and decreased the number of autophagic‐lysosomes in Panc‐1 cells. (b) Western blot detected LC3II/I in TPEN‐treated Panc‐1 cells. (c) The ROS levels of Panc‐1 cells treated with CQ or TPEN was measured by DCFH‐DA probe fluorescence detection, and at the same time, the lysosomal activity was measured by lysotracker in the same cells. All the results are presented as the means ± SEM of values obtained in three independent experiments, *p < 0.05, **p < 0.01, ***p < 0.001 (Student’s t‐test). CQ: chloroquine diphosphate salt; DCFH‐DA: 2′,7′‐dichlorofluorescin diacetate; ROS: reactive oxygen species; SEM: standard error of mean; TPEN: N,N,N,N‐Tetrakis(2‐pyridylmethyl)‐ethylenediamine [Color figure can be viewed at wileyonlinelibrary.com]. The present work demonstrates a cell‐death‐inducing effect of the zinc chelator TPEN on pancreatic cancer cells. As demonstrated here (Figure 8a), we found that the depletion of zinc by TPEN triggered cell death of pancreatic cancer cells via mitochondrial dysfunction and lysosomal disruption, and TPEN‐induced cell death in Panc‐1 cells could be completely reversed by ZnSO4 and partially reversed by NAC. The deregulated zinc transporters have been linked with several cancers (Li et al., 2009). In breast cancer patients, zinc content is increased in tumor tissues and decreased in the serum (Hoang et al., 2016). Our results confirmed the same pattern in pancreatic cancers, where the level of zinc was higher than that in normal tissues. These results demonstrated the zinc dependence in many cancer tissues, which required coordination of zinc transporters to maintain the uptake and utilization of zinc. Previous studies have indicated that several zinc transporters play important roles in regulating cell proliferation and death. For example, many studies indicate that overexpression of ZIP4 confers resistance to apoptosis and silencing of ZIP4 induces apoptosis under zinc‐deficiency conditions in pancreatic cancer (Li et al., 2009). In our experiment, we also demonstrated that the level of ZIP4 in Panc‐1 cells was higher than that in other cancer cells (Figure 8b). And 4 μl TPEN can significantly up‐regulate the expression levels of various ZIPs and ZnTs, but under the condition of 2 μl TPEN, there is no change of ZIPs and ZnTs. These results suggested that 4 μl TPEN induced zinc deficiency in pancreatic cancer cells, which results in responding to zinc deficiency via adjusting the levels of ZIPs and ZnTs (Figure 8c). Although 2 μl TPEN may only stimulate the release of zinc from the “zinc reservoir,” it did not change the overall level of zinc in Panc‐1 cells.KRAS mutation is found in most pancreatic cancer (Agarwal & Saif, 2014; Eser, Schnieke, Schneider, & Saur, 2014) resulted in metabolic alterations, including glutamine metabolism and autophagy activation. Moreover, pancreatic cancer cells efficiently recycle various metabolic substrates through macropinocytosis and autop- hagy (A. Yang et al., 2014). There are reports that show loss of autophagy can induce ROS, leading to apoptosis in pancreatic cancer cells (A. Yang et al., 2014). It is possible that the elevated autophagy may also serve as an adaptation to prevent the cytotoxic ROS accumulation in pancreatic cancer. Inhibition of autophagy by si‐ATG5 in 8988T cells results in an increase of total ROS (A. Yang et al., 2014). Conversely, inhibition of ROS with NAC significantly attenuates autophagy in 8988T cells. These research works indicate that inhibition of autophagy under oxidative stress is very lethal to pancreatic cancer cells. In our study, TPEN not only caused oxidative stress but also inhibited cell autophagy. FIG U RE 8 Schematic diagram of the mechanism that TPEN‐induced cell death. (a) Schematic diagram of the mechanism that TPEN‐induced cell death by increasing ROS and depressing autophagy. (b) Differential expression analysis of zinc chaperone genes in different cancer cells. Semiquantitative analysis confirmed that ZIP4 in Panc‐1 cells was higher than that of other cancer cells. (c) The relative expression of ZnTs and ZIPs in Panc‐1 cells treated with DMSO, 2 µM or 4 µM TPEN was determined by qPCR. Beta‐actin was used as internal standard. The results are presented as the means ± SEM of values obtained in three independent experiments, ***p < 0.001 (Student’s t‐test). DMSO: dimethyl sulfoxide; qPCR: quantitative polymerase chain reaction; ROS: reactive oxygen species; SEM: standard error of mean; TPEN: N,N,N,N‐Tetrakis(2‐ pyridylmethyl)‐ethylenediamine [Color figure can be viewed at wileyonlinelibrary.com]. In conclusion, in this investigation we reported that TPEN induced cell death in pancreatic cancer cells through outbreak of ROS and lysosomal disruption. How did TPEN produce ROS? One explanation we found was that TPEN could stimulate glycolysis and mitochondrial metabolism in a short period of time and decrease GSH production. Meanwhile, TPEN inhibited autophagy through mediating the lysosomal disruption. Therefore, based on the present results, it is proposed that Zinc chelator TPEN should be explored as a potential candidate for PDAC clinical therapy. Of course, the deeply mechanisms of TPEN induced changes in genes and signaling pathways are needed further research. ACKNOWLEDGMENTS This study was supported by the grants from Natural Science Foundation of Heilongjiang Province (ZD2017001), the Fundamental Research Funds for the Central Universities of China (Nos. 2572016EAJ3, 2572016AA14), and the National Natural Science Foundation of China (No. 31472159). CONFLICT OF INTERESTS All authors declare no conflict of interests. AUTHOR CONTRIBUTIONS Zhen Yu wrote the manuscript. Ze Yu and ZhenBao Chen conceived of the study and analyzed data. Lin Yang, MingJun Ma, and ChunSheng Wang participated in the statistical analysis and contributed essential tools and reagents. Ze Yu, ChunBo Teng, and Yu Zhe Nie analyzed the data and revised the paper. 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